Trichophyton extract and method of preparing the same



Patented Nov. 4, 1941 UNITED STATES TRICHOPHYTON EXTRACT AND METHOD OF PREPARING THE SAME Samuel M. Peek, New York, N. Y., assignor to Mount Sinai. Hospital Research Foundation, Inc., New York, N. Y., a corporation of New York No Drawing. Application August 7, 1939, Serial No. 288,763

8 Claims.

The present invention relates to fungus ex tracts and methods of producing them and, more particularly, extracts of fungi produced by the growth of an organism in a suitabl medium.

The invention has especial importance in the production of trichophytin, as well as other bacterial extracts now used in the diagnosis and treatment of certain diseases.

It is known in the literature and for some years has been common practice to use bacterial and fungus products of the foregoing type to diagnose disease. patient intradermally with the extract and observing whether a local reaction (e. g. positive skintest) occurs as a result thereof in 24 to 48 hours. Certain of these substances have also been used to treat, i. e. desensitize against the disease by subcutaneous, intradermal or intramuscular injection of the patient in suitable dosages.

- Care must be exercised in this use of these extracts lest the patient be made worse where his condition is such that he cannot tolerate the injection of such substances.

At the present time, little is known about com- I ponents of these extracts, especially trichophytin,

or what properties or ingredients thereof cause the reactions produced thereby or the immuniza tion 1. e. desensitization frequently resulting from the use thereof.

Trichophytin can be defined as an extract of cultures of single or several species of Trichophyton, the ringworm fungus, used in the diagnosis and treatment of the difi'erent varieties of ringworm.

Trichophytin is .now prepared by inoculating Sabourauds medium (fluid) with a fungus (such as Trichophyto/n, gyp'seum) and permitting it to grow for from ten weeks to three months until This is accomplished by injecting the a large pellicle has formed, covering the surface.

of other organisms, such as staphylococcus (contaminating organisms), the pH value of the medium is maintained at a value that is less favorable to other organisms than the particular organism with which the medium is inoculated. For example, in preparing trichophytin, an extract used in diagnosing and treating skin diseases such as athletes foot, the medium is initiated at a pH value of approximately 6.0 to 6.5. The pH value of the pellicle changes very little and, up to the present time, the pH values of the culture have been disregarded, interest being centered only in the pellicle.

As a result of exhaustive tests, I have discovered that preparations of the type hereinabove mentioned can be formed in vitro' with certain attendant characteristics that can be utilized not only to control their biologic activity and other characteristic properties bearing upon the effect of the extracts, but also the very nature of the extract itself in so far as its component parts are concerned.

An object of the invention, accordingly, is to provide fungus extracts of the type used in the diagnosis or treatment of certain diseases, for example, preparations such as trichophytin, the extracts being characterized by certain useful properties not heretofore available in stances.

A further object of the invention is to provide extracts of the above character, the properties of which are adjusted in a predetermined desired manner.

Yet another object of the invention is to provide extracts of the above type, the component parts or ingredients of which are varied to render them most suitable for use in either the diagnosis or the treatment of disease.

A further object of the invention is to provide a new method of preparing extracts of the above type.

A further object of the invention is to provide a new method of forming extracts of the above type wherein the strength, concentration or activity thereof is 'efiectively controlled.

A further object of the invention is to provide a new method of determining the potential or actual concentration of the extracts or ingredients thereof in vitro.

Yet another object of the invention is to provide a method of the above character wherein the composition of the extract as to its component parts is selectively controlled.

Further objects of the invention will appear as it is described in further detail hereinafter and, for the purposes of this description, the invention will be described in connection with the extract trichophytin and its production although it is to be understood that the invention has equal such 'sube organism used, but with trichophytin, generally around 8.0 to 8.5.

I have found that Trichophyton aypseum, for example, is a very hardy organism and will grow in the aforesaid media at starting pH values from 4 to 10. In every instance, however, the final pH value, after a substantial period of growth, is approximately 8.0 to 8.5. The organism, accordingly, is able to acclimatize itself or change its surroundings to suit itself within this range of starting pH values.

In analyzing this phenomenon and observing the reaction of patients to variously prepared extracts of this type over long periods of time and under varying conditions, I have discovered that certain properties or components are developed when the organism grows in media, the starting pH of which is above 8.0 while others are produced when the starting pH is below 8. I have found that the organism is capable of developing a substance to make the media more acid when the starting pH is above the optimum pH value (8), and is also capable of developing a substance or substances to render the media more alkaline when the starting pH is below the optimum value of approximately 8.

It has been known for some time that patients who have developed an infection from fungi become hypersensitive to the organisms or their products. This sensitivity very often results in disease manifestations which on the skin are known as trichophytids, and in fact it is very well agreed among workers in this field that the disease process itself is due to the results of the direct invasion of the organism by the pathogens plus the reaction of the sensitized tissue when this sensitivity to the organisms or their products has developed. Their allergic or hypersensitivity reactions when due to fungi are known as trichophytids.

As a matter of fact as far as fungus infections such as athlete's foot are concerned the main pathogenic manifestations are due to the trichophytids. Once this hypersensitivity has developed it can be recognized by the intradermal injection of trichophytin. In a specifically hypersensitive individual there will be produced in 24 or 48 hours an inflammatory reaction at the im'ection site which is then called a positive trichophytin test.

My further studies and tests have led to my discovery that the skin reaction of hypersensitivity, may be produced by making a trichophytin with a saline extract of the pellicle itself washed free of any of the fluid media and its contents, instead of utilizing the specific soluble substance which the trichophyton produces in the fiuid medium.

The organism itself is the pellicle heretofore alluded to in describing the present method of forming the extracts. I have found that it is unnecessary to await its devolopment to the advanced stage of forming a heavy pellicle over the media (usually 10 weeks), as has heretofore been thought necessary. On the contrary, I have discovered that this greater or more advanced growth of the pellicle does not of necessity increase the concentration of the specific soluble substance in the extract. This growth merely provides a larger quantity of the organism and I have discovered that an equivalent quantity obtained by accumulating numerous pellicles resulting from the growth of the organisms for a shorter time in numerous media is capable of producing an equally active extract when the pellicle alone is used in the preparation of trichophytin.

0n the other hand, I have discovered that the characteristics of the soluble specific substance of trichophytin formed in the medium depend upon two factors, viz., the age of the culture to a minor degree and the method of preparing or buffering the culture media to a major degree. For example, I have discovered that the concentration or activity of the specific soluble substance present in the media can be deduced by the change in the pH of the media itself. If the original pH of about 6 is used as the initial pH value of the culture, as is usual, the strength or activity of the specific soluble substance changes very slowly, if at all, once the optimum pH of 8 is reached. It is for this reason that the cultures must be kept for long periods of time in order to produce an adequate trichophytin. This is a highly important feature of my invention inasmuch as it affords an accurate and effective way of measuring or determining the strength or activity of a bacterial filtrate for testing and treatment of disease and saves time in the preparation. By using the hydrogen ion concentration of the media as an indication of the potential or actual concentration of the specific soluble reactive substance, this component of the extract can be effectively regulated in the preparation of trichophytin etc. in accordance with my invention.

Moreover, I have found that the concentration, strength or activity of the specific soluble substance varies with the conditions under which the organism grows. For example, if the organism is forced to grow under more adverse conditions of pH, the strength or activity of the specific soluble substance which it develops will be greater than if the organism is caused to grow under more favorable conditions. This discovery and application thereof to the preparation of extracts therefor, constitutes a further and highly important feature of my invention.

Thus, in present methods heretofore utilized, by starting at a pH value of around 6 and developing the culture, the same kind of specific soluble substance is always produced. I have found that I can develop what appears to be a different kind or type of specific soluble substance. That is, by broadening the base of the starting point for growth of the culture media to a range of from 4 to 10, all possible pH values and ranges are covered that the fungus will meet on the surface of the skin. As there are apparently different kinds of specific soluble substance developed by the organism under different pH values, I therefore am able to get every possible type of desensitizing substance that can be developed.

In determining comparative strengths of extracts formed under varying conditions, I found that with an initial pH of 10 the change to the optimum value is too rapid. That is, the substance that the organism produces to render the medium more acid under these conditions apparently is developed very rapidly and without causing the organism to exert itself to a very great extent.

In order to determine the difference between the concentration and type of specific skin reaction to substances developed by the organism to render the media more acid and to render the media more alkaline, I inoculated the medium with Trichophyton gypseum at a starting pH of 10 and at a starting pH of 4. Samples of pellicles Q making trichophytin at a starting value of pH 6.5 are used and when the trichophytin is made (10-15 weeks) the final pH is 8.0 or 8.5. In all,

there was a rise oil and pH value. When trichophytin is prepared at a starting pH of 4 and a rise of value to pH of 6 is noted even though the culture is seven to ten days old, there is as much specific soluble substance formed in the culture media as there is in a ten to twelve week old trichophytin extract formed at a starting value. of 6.5. In fact a rise of 2 pH values starting from a pH of 4 indicates already a biologically strong trichophytin; as strong or more active than a ten to twelve week old trichophytin prepared in the old manner from a starting pH of 6.5 containing both pellicle and fluid media as described previously. The strength or concentration of the specific. soluble substance increases with the rise in the pH value until the optimum pH of 8 is reached.

It has been further found that there is more specific soluble substance formed when starting at a pH of 4 to 6 than when the actual pH value is 6 and there is a rise of 6 to 8.

When the initial starting value is 10 there is a quick reduction of the pH value (acidification) to 8 but the amount of specific soluble substance is much less than that produced from 4 to 6.

It has further been found that when the initial pH of 10 is produced by the addition of phosphate buffers (McIllvanes) a different mechanism for production of the specific soluble substance is brought about. Instead of the original pH of 10 going down to 8, the pH value will drop below 8; sometimes to as low as and then gradually build up to 8 again where the pH is maintained.

In using various trichophytin fractions on patients in testing their hypersensitivity to trichophytin, I found in those fractions which were taken when the pH value of the media was dropping from a pH of to below 8, i. e. 5 to 6, there was very little skin reaction after intradermal injection even in suitable patients.

In the treatment of patients with trichophytids the same extracts used for diagnosis are also used for treatment or desensitization. After adequate injections, especially with trichophytin, the patient is desensitized so that he finally does not give an intradermal test to trichophytin. This is usually accompanied by a marked amelioration of symptoms -i. e. disappearance of trichophytids. In treatment, furthermore, one has to be very cautious in judging dosage as too great a dose will cause focal reactions or even generalization of the disease.- If, therefore, an extract can be made that will desensitize and yet not give any or practically no local reaction one could desensitize or immunize with no danger. In other words, in such a conception one indicates that there are two factors. a skin sensitizing factor A which is found in the pellicle and in the media and is designated as specific soluble substance and another factor B which desensitizes or immunizes and yet gives practically no local skin reaction. This B substance free, or practically free from A. is found only in the medium under suitable conditions. This separate substance which I have termed B is also found in all fungus extracts no matter where the starting point but is accompanied by the A substance. An extract which is having been properly buflered.

capable of desensitizing without causing very much of a local reaction is only found in media I have discovered that the specific soluble substance which is one ingredient of the extract-is produced most effectively when the organism grows in media at a starting pH value of 4.0 which increases with the growth of the organism, whereas the B substance which is another ingredient of the extract is produced relatively free from A only when the organism grows in media at a starting pH value of above 8, usually 10, especially if a phosphate buffer is added to the media.

It appears that. with a starting pH value above the optimum value of 8, and with a medium that is rendered alkaline, for example, by sodium hydroxide, the organism will cause the pH value of the medium to rise and fall periodically .but with a mean decrease in the pH value until the optimum is reached. In order to cause a drop in the pH below 8 with a starting point of 10 as has been indicated, I have found that McIllvanes buflers (phosphate) have to be used. I have found that the change from a pH of above 8 on the drop even with the use of phosphate buffers is so rapid that very-often even with daily pH reading the point of crucial taking of a sample 1. e. only on the drop, especially from 8 to 6 is missed or not enough of B alone is made to be of practical value. This is especially important since as soon as the tide swings upward again A appears in quite marked concentration. In order to buffer the media more efi'ectively and control the resultant pH, I have found that by using human blood with the media instead of the phosphate buffer previously referred to, the pH value of the media decreases steadily to a minimum value well below the optimum value of 8 and that this decrease is maintained for a relatively long time so that very little specific soluble substance is produced and there is adequate time to take samples. A substance or extract prepared with a great deal of B and very little A will bring about immunization or desensitization with practically no local or generalized reaction possible. I have found that the pH value of the media under the foregoing conditions remains at the minimum value for a considerable time and then gradually increases to the optimum pH value of 8. During this increase, I find that the specific soluble substance is produced. I have thus discovered an effective way of enabling extracts to be prepared which will serve to desensitize without, at the same time, subjecting the patient to the possibility of a reaction and which, under certain conditions of the patient, becomes a very dangerous procedure.

By using the blood serum as a bufier for the medium, much better results are obtained not only when the starting pH of the media is above the optimum value, but also when the starting pH is below the optimum value. Blood serum thus can be used to slow up the changes in the pH value in the media and also to produce more of the A and B in a given time. When blood serum is used at a starting pH value of 4 it also slows up the gradual rise of the pH to the optimum value of 8 and thus in a given unit of time a greater amount of A is formed since it is ap- 4 fight a buffer to produce its optimum pH value.

In a similar manner blood acting as a bufier at showing that the patients are sensitive.

a starting pH value of 10 also causes the organism to work harder to bring about its optimum pH value and thus it produces so much of B and A, but mostly B that it overshoots the mark and carries it over to 5 or 6.

I have also found that the production of the B substance cannot be controlled when veronal is used as the buffer, although veronal can be used for buffering the organism in media having both high and low starting pH values.

To illustrate the effectiveness of the serum prepared in accordance with the present invention, I have tested patients with trichophytin that is available on the market and obtained reactions I have then treated such patients with the presently available trichophytin until they show no test or reaction, thus apparently indicating that the patients were desensitized but still showing trichophytids. However, I have then tested these patients with trichophytin prepared in accordance with this invention (especially fractions prepared from pH 4 to 8 and buffered) and have found that the patients still reacted to trichophytin showing that they are still sensitive. Therefore they have not been effectively treated because there was an inadequate amount of specific soluble substance and desensitizing substance found in the commercial extracts. It follows that patients desensitized with ordinary trichophytin do not do as well clinically as those treated with my extracts.

It will thus be observed that, as a result of my invention, I am able to prepare fungus extracts either with or without the specific soluble substance which is the ingredient that causes a reaction in the patient showing that the patient is sensitive specifically. By producing extracts having none or very little specific soluble substance, but a great deal of desensitizing substance I am able to treat effectively patients who are already suffering from the disease without producing local or generalized reactions.

Moreover, the invention affords an effective method of determining the activity of an organism filtrate for the testing and treatment of disease by using the hydrogen ion concentration of the media within which the organism grows as an indicator for the potential or actual concentration of the specific soluble reactive substance.

The invention further provides an effective method of buffering the organism to increase the concentration of specific soluble substance.

The invention further provides a selective method of buffering the organism to develop, selectively and either individually or simultaneously, the specific soluble reactive substance and the desensitizing or immunizing substance. This method utilizes the control of the initial hydrogen ion concentration of the media and the proper use of a buffer, to provide the desired result.

While the invention has been described specifically in connection with trichophytin, it is equally applicable to the preparation of other substances.

I claim:

1. The method of preparing and controlling the potency of a trichophyton extract comprising preparing a culture of Trichophyton gypseum, the initial pH value of which is greater than the optimum pH value developed by growth of Trichophyton gypseum, which Trichophyton gypseum, upon decrease of pH value from above the optimum, develops an immunizing or desensitizing substance mainly, and upon increase of pH value from below the optimum develops both reactive and immunizing substance, permitting the Trichophyton gypseum to grow, to cause the pH value thereof to decrease continuously, and utilizing the culture before the pH value thereof increases to produce an extract.

2. The method of preparing a trichophyton extract for immunizing or desensitizing against fungus infections comprising preparing a culture of Trichophyton gypseum, the pH value of which is greater than the optimum pH value developed by growth of the Trichophyton yypseum, which Trichophy'ton gypseum, upon decrease of pH from above the optimum develops immunizing substance mainly and upon increase of pH from below the optimum develops reactive and immunizing substance, introducing an agent to buffer the culture, permitting the Trz'chophyton gypseum to grow to cause the pH value thereof to decrease continuously and utilizing the culture before the pH value thereof increases to produce an extract.

3. The method of preparing a trichophyton extract for immunizing against fungus infections, comprising preparing a culture of Trz'chophyton gypseum, the pH value of which is greater than the optimum pH value developed by growth of the Trichophyton ypseum, which Trichophyton yypseum upon decrease of pH develops immunizing substance mainly, and upon increase of pH develops reactive substance and immunizing substance, introducing blood serum to buffer the culture, permitting the Trichophyton gypseum to grow to cause the pH value thereof to decrease continuously, and utilizing the culture before the pH value thereof increases to produce an extract.

4. The method of preparing a Trichophyto gypseum extract for immunizing against fungus infections, comprising preparing a culture of Trichophyton gypscum, the pH value of which is greater than the optimum pH value developed by growth of the Trichophyton gzmseum, which Trichophyton yz/Pseum upon decrease of pH develops immunizing substance mainly, and upon increase of pH develops reactive substance and immunizing substance, introducing a phosphate buffer to buffer the culture, permitting the Trichophyton gypseum to grow to cause the pH value thereof to decrease continuously, and utilizing the culture before the pH value thereof increases to produce an extract.

5. A Trichophyton oypseum extract having a predetermined controlled potency and having chiefly immunizing properties and substantially no skin reactive factors, prepared by the method set forth in claim 1.

6. A Trz'chophyton ypseum extract having a predetermined controlled potency and having chiefly immunizing properties and substantially no skin reactive factors, prepared by the method set forth in claim 2.

7. A Trichophyton gypseum extract having a predetermined controlled potency and having chiefly immunizing properties and substantially no skin reactive factors, prepared by the method set forth in claim 3.

8. A Trichophyton gypseum extract having a predetermined controlled potency and having chiefly immunizing properties and substantially no skin reactive factors, prepared by the method set forth in claim 4.

SAMUEL M. PECK. 

